UNIVERSITI SAINS MALAYSIA
MAY 2010Dissertation Submitted In Partial Fulfillment Of TheRequirementsFor The DegreeOf Masters OfPathology
(Haematology)
DETECTION OF IRON DEFICIENCY ANAEMIA BY RETIC HAEMOGLOBIN (RET-He) IN HAEMODIALYSIS PATIENTS ON RECOMBINANT HUMAN
ERYTHROPOIETIN (rHuEPO).
BY
DR EHRAM B JAMIAN M.D. (UNIMAS)
ffi KI
UNIVERSITI SAINS MALAYSIA
i
ACKNOWLEDGEMENT
All praises to Allah s.w.tthemost Merciful andthe most Benificient.
Firstly, I would like express may thankfulness to Allah s.w.t for giving me the strength and ability to complete this researchproject. In theprocess, I am indebted to many people for their assistance. With that, I would like to express my greatest appreciation to every individual who wereinvolved inmakingthis project a success.
With great pleasure, I would like to acknowledge Assoc. Prof. Dr Rosline Hassan, my supervisor and Head of Haematology Department, PPSP for her continous supervision, ideas and support. I am grateful to Dr Marini Ramli, my co-supervisor and lecturer of Haematology Department, PPSPfor herencouragement.
I also wish to thank Assoc. Prof. Kamaliah Bt Mohd Daud andDrShukeri Bin Mohamad
Abdullah for their technical support and laboratory direction of this project. This study
I would also like to thank my respected parents, Dato’ Hj Jamian Palil, Datin Hjh Kartini Yahya and my brother En Ahmad Zaki, for their love, understanding and sacrifices throughout my study.
ii was supported byshort term grant 304/PPSP/6131627 from Universiti Sains Malaysia.
as my co- supervisors. Special thanks go to En Abu Salam and Puan Suryati Bte
TABLE OF CONTENTS Contents Page
1. Title i
2. Acknowledgement ii
iii -iv 3. Table ofcontents
4.List of tables v 5. List of figures vi
vii - viii 6. List of abbreviations
7. Definition ix
x - xi 8. Abstrak( in Malay language )
xii- xiii 9. Abstract ( in English)
10. Chapter1 : GeneralIntroduction 1-3 11. Chapter 2 : Literaturereview
2.1 Iron Metabolism
2.2 Erythropoiesis And Iron Deficiency
2.3 AnaemiaIn ESRF Patient ( Functional Iron Deficiency ) 2.4 Iron StatusAnalysis
12. Chapter 3 : Objectives of the Study 3.1. General objective
3.2. Specific objectives 13. Chapter 4 : Methodology
4.1. Studydesign 4.2. Sampling forcases 4.3 Sample size calculation 4.4 Laboratory methods 4.5 Data entry
14. Chapter 5 : Results 5.1 Characteristic Data
5.2 Detection ofiron deficiency anaemiaby ferritin,sTIR and
24 24-27 29-30 31-39
39
40-42 43 23 23 4-7 8-9 10-15 16-22
44-45 46 47
52-53 54-70 15. Chapter 6 : Discussion
16. Chapter7 : Conclusion 71
72-78 17. References
18. Appendices
19. Presentation from this research
iv 48-49
50 51 RET-He.
5.3 Analysis of RBC indicesand haematology parameters in IDA and non IDA patients detectby RET-He.
5.4 Correlation between RET-He in IDA group with other haematologicalparameters.
5.5 Agreement between RET-He with sTfR, ferritinand TfR-Findex in detection of iron deficiency anaemia.
5.6 Detection of IDA by diagnostic plot using RET-He and TfR-F index.
5.7 Assessment oftreatmentresponse in IDA patients by RET-He 5.8 Therapeutic implications of the diagnosticplot afterthe iron
treatment
5.9 Sensitivity, specificity and AUC of thestudied iron parameter
LISTOF TABLES
StatesOfIronDeficiency Table 2.1
Characteristic data of the patients.
Table 5.1
Haematological findings in theESRF patients.
Table 5.2
Detection of iron deficiencyanaemiabyferritin, sTIR and RET-He Table 5.3
Table 5.4 Comparison haematological parameters of IDA in hemodialysis patients variousaccording to RET-He.
Table 5.5 Correlation between RET-He in IDA group with other haematological parameters.
Table 5.6 Agreement between RET-He with sTfR, ferritin and TfR-F index in detecting iron deficiencyanaemia.
Table 5.7 Detection of IDAby diagnostic plot using RET-He and TfR-F index.
Table 5.8 Responded to iron treatment in IDA patients by RET-He Table 5.9 Therapeutic Implications of theDiagnostic Plot
Table 5.10 Sensitivity, specificityand AUC ofthe studied ironparameter
LISTOF FIGURES
The interplay of iron and erythropoietin inerythropoiesis.
Figure 2.1
Thomas Diagram Figure 2.2
Flow Chartof Research Methodology Figure4.1
DetectionofIDAbyRet-He(Gendercomparison) Figure 5.1
ThomasDiagram Figure 5.2
ROC Curve Iron Parameter Figure 5.3
ThomasDiagram Figure 6.1
Thomas Diagram (Diagnosticand Treatment Plot) Figure6.2
Thomas Diagram (Post Iron Treatment Plot) Figure 6.3
vi
LISTOF ABBREVIATIONS
Anaemia Of Chronic Disease ACD
C reactiveprotein CRP
Divalent MetalTransporter!
DMTI
Deoxyribonucleic Acid DNA
End Stage Renal Failure ESRF
Ferrous
Ferric
FunctionalIron Deficiency FID
Haemoglobin Hb
Haemodialysis HD
HospitalRajaPerempuan Zainab HRPZ
Iron deficiency anaemia IDA
Mean Cell Haemoglobin MCH
MCV Mean Cell Volume
rHuEpo recombinantHuman Erythropoietin Fe2+
Fe3+
Reticulocyte Hemoglobincontent RET-He
Receiveroperating characteristic curve ROC
Red cell distributionwidth RDW
SolubleTransferrinReceptor sTfR
Total iron bindingcapacity TIBC
Transferrin Saturation TSat
Zinc Protoporphyrin ZPP
viii
DEFINITION
Classical iron deficiency anaemia
Conventional methodforiron
Functional IronDeficiency FID
Ironrestricted erythropoiesis
Iron replete Ironstore present.
Iron deplete Iron store reduced markedly or absent Nutritional anaemia Anaemia due to iron, B12 and Folate
deficiencyor due to alcoholism.
Condition whereby iron storedepleted, low serumiron but normal or low hemoglobin.
Imbalance between the iron requirement of the erythroidmarrow and the actual iron supply.
Method such as serumiron,
transferrin/total iron binding capacity, transferrin saturation and ferritin.
Depletionofstorage and functional iron compounds.Decreased haemoglobinization of red cells.
ABSTRAK
Kekurangan zat besi sering berlaku di kalangan pesakit buah pinggang peringkat akhir yang diberikan hormon erithropoitin (EPO) sebagai rawatan. Kekuranganzat besi adalah faktor utama yang menyebabkan pesakit tidak bertindak balas dengan baik dengan rawatan hormon tersebut.
Tujuan utama kajian ini adalah untuk menentukan ketepatan penggunaan RET-He (Retikulosit Hemoglobin) dalam mengenalpasti kekurangan zat besi di dalam pesakit hemodialisis yang diberikan hormon (EPO) berbanding parameter konvensional (ferritin dan penerima transferrin). Selain itu kajian ini juga diharap dapat mengenalpasti perubahan RET-He semasa pesakit diberikan rawatan hormone EPO serentak dengan pemberian zat besi, sama ada parameter ini berupaya menganalisa kadar zat besi yang mencukupi kepadapesakit.
Kajian ini telah dijalankan di HRPZ II bermula dari November 2008 hingga November 2009. Lima puluh lima sampel darah telah diambil dan dianalisa untuk RET-He, ferritin dan penerima transferrin. RET-He dianalisa oleh mesin XE-2100 (Sysmex) Ferritin pula oleh kaedah ELISA(AXSYM) dan penerima transferrin oleh kaedah ELISA (Biovender).
Data akan dianalisa oleh program SPSS version 12.0.
x
Keputusan menunjukkan RET-He mempunyai signifikan dengan penerima transferrin, ferritin, MCV, MCH dan Indek ferritin ( p<0.01 ) Min RET-He di dalam pesakit hemodialisisialah 29.91±2.29 pg. Sensiviti danspecificity RET- He pula ialah 78.3% dan 92% bersama kawasan di bawah kecerunan 0.864. Peratusan pesakit kekurangan zat besi yang dapat dikesan oleh RET-He adalah 63.64%, oleh penerima transferrin 3.64% dan terhadap penerima ferritin 0%. RET-He mempunyai hubungan yang signifikan
transferrin r= 0.042 dan indek ferritin r=0.036, walaubagaimanapun tiada hubungan yang signifikan terhadap ferritin. Kajian seterusnya menunjukkan pesakit yang dikesan mengidap kekurangan zatbesi dan responterhadap zatbesi yang diberikan dapat dikesan dengan baik olehRET-HE ( p<0.01 )
Sehingga kini pengesanan kadar kekurangan zat besi di kalangan pesakit hemodialisis rendah.
adalah konvensional
oleh kaedah diberikan hormon EPO
yang
Walaubagaimanapin kaedah yang terkini oleh RET-He menunjukkan bahawa ia adalah sensitif danspesifik parameter untuk analisa status zat besi di dalam pesakit hemodialisis yang diberikan hormone EPO.
Oleh yang demikian kami mencadangkan penggunaan RET-He sebagai parameter utama untuk diagnosis kekurangan zat besi dan juga sebagai kaedah monitor tindak balas kepada rawatan intravenous zat besi kepada pesakit hemodialisis .
ABSTRACT
Iron deficiency anaemia (IDA) appears frequently in haemodialysis (HD) patients with rHuEPO (recombinant human erythropoietin) therapy. Iron deficiency is the most
indices are inadequate to demonstrate the status or utility of iron in erythropoiesis. The aim of this study was to clarify the accuracy of RET-He in diagnosing IDA in haemodialysis patients with rHuEPO therapy compared with conventional iron parameters. Secondly to detect the changes in RET-He during iron supplementation for IDA patients, either this marker isa prospective and reliable indicatorof iron sufficiency.
A cross sectional study was done atHRPZ II from November 2008 to November 2009.
55 samples was collected and analysed for RET-He, ferritin and sTIR. RET-He was measured with the XE-2100 (Sysmex). Ferritin values were determined using ELISA method by AXSYM and sTfR by ELISA Biovender. Data were analyzed using SPSS software version 12.0.
RET-He was significantly correlated with sTIR, MVC, MCH and TfR-F index 29.91+2.29 pg. Sensitivity and specificity of RET-He, 78.3% and 92 % respectively with AUC of 0.864. Percentage of IDA detected by RET-He was 63.64% but by sTfR and ferritin were 3.64% and 0%
respectively. RET-He was significantly had aggrement with sTfR and TfR-F index, r=
0.042andr=0.036 respectively, however noagreement with serum ferritin.
xii (p< 0.01). Mean RET-He in haemodialysis patients was
common factor associated with rHuEPO hyporesponsiveness. However current iron
supplement can be detectvery wellby RET-He (p< 0.01).
To date detection rate ofIDA in haemodialysis patients with rHuEPO by conventional method (sTfR & ferritin ) was low. However our current method by RET-He shows that it isasensitive and specificmarker of iron status in haemodialysis patients withrHuEPO.
well as monitoringof responses tointravenous irontherapy in haemodialysis patients.
Thus we strongly suggest using RET-He as adiagnostic tool for the detection of IDA as A follow up study showed those who were diagnosed had IDA respond to iron
